Modified cytosine residues in DNA show asymmetric distribution within gene bodies, wherein 5mC, and its oxidized derivatives 5-hydroxymethylcytosine (5hmC) and 5-carboxylcytosine (5caC), are enriched at exons of actively transcribed genes. Combined with evidence that spliceosome assembly on nascent mRNA occurs co-transcriptionally, methylation thus has the potential to directly or indirectly influence splicing decisions. We are exploring these possibilities with a focus on the specific contributions of distinct cytosine modifications. To this end, we utilized knockout mouse embryonic stem cells (mESCs) in which the enzymes that generate cytosine modifications in DNA are selectively deleted. The impact of altered methylation on specific modes of pre-mRNA splicing has been assessed and targeted methylation will be performed at select events to establish the direct influence of methylation on splicing and to further query the mechanistic underpinnings. In addition, to these general investigations, we are studying the indirect effect of 5mC on pre-mRNA splicing through modulation of CTCF-dependent splicing. We previously showed that CTCF and 5mC mediate reciprocal effects on alternative pre-mRNA splicing, wherein CTCF promotes spliceosome assembly at weak upstream exons through local pol II pausing, while overlapping 5mC acts to evict CTCF and is thus associated with exon exclusion (Shukla et al., Nature, 2011). We subsequently showed that coordinated exchange between CTCF and 5mC in regulated alternative pre-mRNA splicing is mediated by the TET proteins (Marina et al., EMBO J, 2016), and further found that CTCF binding is enhanced in the presence of overlapping 5caC as compared to unmethylated DNA. Building on this observation, we are exploring the mechanistic basis for augmented CTCF interaction with 5caC-containing DNA and are examining for unique features in the associated impact on splicing. Co-transcriptional splicing further creates an opportunity for the spliceosome to influence intragenic chromatin. We thus explored whether the spliceosome plays an instructive role in targeting DNA methylation to exons. Current mechanisms for intragenic methylation involve direct recruitment of the de novo methyltransferase DNMT3b through association with trimethylated H3 lysine 36 (H3K36me3), a hallmark of active transcription. However, DNA methylation is not evenly distributed to H3K36me3-rich regions, suggesting additional levels of regulation. Based on specific detection at the exons of spliced genes in organisms with overall lower methylation, we tested the direct involvement of splicing in the distribution of genic methylation. To this end, we generated isogenic cell lines for inducible expression of minigene-derived RNA in the presence or absence of competent splice sites. The resulting analysis showed that the ability to acquire or maintain targeted methylation is unaltered in the presence of competent splicing signals (Nanan et al., Nucleic Acids Res., 2017). Based on emerging evidence implicating modified ribonucleosides in mRNA stability and translation, we recently expanded our investigations to include cytidine modifications in mRNA. Of the three cytosine base modifications that have been detected in mRNA, 5-methylcytidine (m5C) and 5-hydroxymethylcytidine (hm5C) are direct analogs of 5mC and 5hmC in DNA and have been explored through existing reagents. In contrast, the singular acetylation event in RNA, ac4C, remained uncharacterized. To examine the occurrence and function of mRNA acetylation, we performed transcriptome-wide mapping of ac4C and associated RNA-sequencing in wildtype cells, and cells depleted of the putative acetyltransferase NAT10. Our results clearly demonstrated that ac4C promotes expression of its target mRNAs through enhancing translation (Arango et al., in revision). Future studies are aimed at examining ac4C regulation in distinct cellular conditions, as well as the mechanism by which ac4C impacts translation.